Process for obtaining chloroamphenicol



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` Serial lvm- 15,265

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ma i y amI negatwezbaeteria The new antibioticv has been found to givetherapeutic blood levels when administered by either the parenteral ororal routes. The fact that it exerts its beneficial effect whenadministered orally is surprising in view of the fact that at thepresent time penicillin is almost the only antibiotic which givestherapeutic blood levels on oral administration. Even in this case thedestruction of the penicillin by the-digestive fluids renders itnecessary to administer approximately three to fourtimes theintramuscular quantity of penicillin in order to obtain therapeuticblood levels. The antibiotics which are most eective against gramnegative bacteria, such as streptomycin, are completely inactive whenadministered orally due to the fact that they are not absorbed from theintestinal tract. Thus it will be appreciated that this new antibioticrepresents the rst antibiotic exhibiting a high degree of activityagainst gram negative bacteria which is therapeutically effective uponoral administration. The usual oral or parenteral dosage ofchloramphenicolin the treatment of urinary or gastro-intestinal tractinfections is one t`o ten grams daily depending, of course, upon thetype and extent of the infection.

Microbiological method for preparing 4(l)\,l11o

nitrophenyl-Z-dichloroacetamidopropane-I, 3- dol The new antibioticcompound, chloramphenicol,(l)-ip-l-p-nitrophenyl-2-dichloroacetamidopropane- 1, 3diol, may beprepared from cultures of an Actinomycete called Streptomycesvenezuelae. This microorganism occurs inter alia in soils and ischaracterized by branched slender mycelium rarely or not septate, aerialhypae giving rise apparently endogenously to chains of unicellularspores not normally fragmenting as oidia, unbranched sporophores andspore chains. Cultures of this organism may be obtained by mixingcultures of the specific bacteria inhibited by the micro-organism withaqueous agar and adding a soil containing the desired Streptomycesvenezuelae. After incubating the mixture for one to ten days colonies of.the desired Actinomycete and other antagonists appear. TheStrepto'myces venezuelae growths are selected, transferred to a freshnutrient medium and later isolated as a pure culture in accordance withthe conventional procedures. Y

According to the invention(1)-1/1-1-p-nitrophenyl-2dichloroacetamidopropane 1,3-diol is producedby inoculating a suitable nutrient me'- dium with Streptomycesoenezuelae and incubating the mixture under aerobic conditions at aboutto 40 C. for about two to fifteen days. After removal of the solidmaterial present in the culture mixture, the desired antibiotic isisolated from the culture liquid.

The cultivation of the organism in the aqueous nutrient medium may becarried out in a number of different ways. For example, themicro-organism may be cultivated under aerobic conditions on the surfaceof the medium or it may be cultivated beneath the surface of the medium,i. e. in the submerged condition, if oxygen is simultaneously supplied.

temperature (about C.) for a. period of about ten to fifteen days. Themvcelium is then removed from the liquid containing the desired abouttwo to seven days. Under these conditions Briefly stated, the productionof this new antii biotic by the surface culture methodi'nvolvesinbetween about `20 and 40 C., preferably at room the organism developsas numerous more or less discrete particles dispersed throughout themedium in contrast to the more or less continuous pellicle present onthe surface of the medium in the surface culture method. By virtue ofthis distribution of the organism throughout the medium, large volumesof the inoculated nutrient medium can be cultivated at one time in thelarge tanks and vats customarily employed in the fermentation industry.Stationary vat fermenters equipped with suitable agitation and aerationdevices as well as horizontal rotary drum fermenters have been found tobe particularly useful in this respect. However, for the preparation ofsmaller quantities of the antibiotic or of cultures of themicro-organism this submerged culture method `through open pipes,perforated pipes, porous diffusion media such as carbon sticks.carborundum, sintered glass and the like, or it may be provided byspraying, splashing or spilling themash into or through anoxygen-containing atmosphere.

A wide variety of nutrient media may be used in the growing stage of theprocess. However, it has been found that the best results are obtainedwhen an aqueous medium containing an assimilable carbon source and aproteinaceous material is employed. Assimilable carbon source is hereunderstood to include polyhydric alcohols and mono, di, andpoly-saccharides while the term proteinaceous material includesunmodified pro- I tein and protein degradation products, particularlysuchproducts as arise from the hydrolysis of proteins. These porteindegradation products include proteoses, peptones, polypeptides, peptidesand amino acids.

As assimilable carbon sources glycerol, sorbitol, glucose, fructose,sucrose. inulin, dextrins and starches may be mentioned. These carbonsources may be used in purified form or in the form of concentrates suchas whey concentrates, liquefied or modified starches, corn steep liquor,molasses, corn syrup, grain mashes and the like. Some of the grainmashes, as well as some whey concentrates, are sufiiciently `rich inprotein that extra proteinaceous adjuncts need not be added to themedium. These latter substances also contain an appreciable quantity ofminerals and Y growth factors which are favorable for the production ofthe antibiotic. Sugar alcohols, and in particular, glycerol, have .beenfound to be very desirabieas constituents of the medium since theyappear to provide a portion 'of the antibiotic in aform readilyaccessible for reaction and thus increase the amount of the antibioticwhich can be obtained from a given medium.

Some examples of the proteinaceous materials which may be used in thenutrient medium are: acid or enzyme hydrolyzed casein, distillers grainslops, distillers solubles, corn or wheat steep liquor. whey or wheyconcentrates. soybean meal, acid hydrolyzed corn or wheat gluten,peptone, brewers yeast, oials and synthetic mixtures of amino acids.These proteinaceous materials need not be supplied in purified form asthe less pure materials which contain traces of growth factors andconsiderable quantities of mineral'nutrients are suitable fr use. Amixture of peptone or amino acids with dried distillers grain slops ordistillers solubles is particularly suitable for the vproteinaceousmaterial of the medium.

I have found that the isolation of the pure i crystalline antibioticfrom the culture medium can be accomplished in a number of diierentways. However, in general, my preferred process comprises clarifying thecrude culture liquid below about pH 10; extracting the solution with anorganic solvent having a solvent-water distribution coefficient for theantibiotic over about 9 such as cyclohexanone, butanol, ethyl acetateand methyl isobutyl ketone; removing the organic solvent fr om theextract; extracting the residue with an organic solvent having asolventwater distribution coeilicient for the antibiotic of betweenabout 1 and 9 such as nitrobenzene, nitromethane, diethyl ether andethylene dichloride; washing the extract with dilute mineral acid andwater or, alternatively, wherea solvent such as a lower dialkyl ether(diethyl ether and the like) or ethylene dichloride has been used as theextraction solvent, passing the extract over an aluminum oxide typeadsorption column; adding water to the organic'solvent extract;evaporating the organic solvent from themixture; extracting the aqueoussolution with an organic fat solvent in which the antibiotic issubstantially insoluble such as benzene or petroleumether; evaporatingthe aqueous solution to the point of 6 separates and concentrating theaqueous `solution to the point of crystallization.

The invention is illustrated by the following examples.

Example 1 A mixture consisting of 180 g. of glycerol, 90 g. of peptone,90 g. of dried distillers grain slops. 90 g. of sodium chloride andsuiiicient distilled water to bring the volume to 18 liters is placed ina 30 liter glass stationary vat-type fermenter provided with a stainlesssteel head and propeller-type agitator. The fermenter also con` tainsvertical baille plates and, near the bottom, a perforated circular airdiffusion ring.

The pH of the nutrient' medium is adjusted to about 7.5 with sodiumhydroxide solution andy the fermenter placed in an autoclave. .'Thefermenter and the medium are sterilized by steam at 120 C. for one hour,the fermenter cooled and then removed from the autoclave. The medium isinoculated with about 900 cc. of a shaken flask culture of Streptomycesvenezuelae, prepared by inoculating 100 cc. portions of a sterilenutrient medium, such as that described above, with spores of the fungusand incubating the mixture in 500 cc. Erlenmeyer flasks on a revolvingshaking machine for about seventy-two hours at room temperature.

After inoculation, the culture mixture is incubated at 24 to 25.5 C. forsixty-five to seventy hours. During the incubation sterile air is passedthrough the diffusion ring into the medium at the crystallization andrecovering the crystalline antibiotic from Athe solution. When the acidextraction method is used, it is preferable to also extract the organicextract with a dilute alkali such as sodium bicarbonate in order toremove acidic impurities.

A modication of the above process consists in eliminating the rstextraction of the clarified culture liquid with an organic solvent andthe subsequent evaporation of the solvent from the extract. In thismodiiied process the clarified culture liquid is extracted with anorganic solvent having a solvent-water distribution coeillcient for theantibiotic of between about 1 and 9 and the remainder of the processcarried out as` described above.

Another process which can be used to isolate the desired (l)-lp1-p-nitrophenyl-2-dichloroacetamidopropane-1,3diol from the clarifiedculture liquid involves adsorbing the antibiotic on activated charcoal'and eluting it with ether orA 80% acetone. Theantibiotic is 'isolatedfrom the eluate by adding water, distilling off the organic Microgramsof k Antibiotic per cc. of Culture Mediuniw Incubation Period, hours Thedesired (l) -p1-p-nitrophenyl-2-dichloroacetamidopropane1,3diol isisolated from'the Y crude culture mixture `or beer containing 132micrograms of the antibiotic per cc. in the foilowing manner:

The crude beer is acidied to pH 2 with dilute hydrochloric acid and thentreated with about 1050 g. of an aluminum silicate filter aid. Afterstirring for one-half hour to an` hour about an additional 1050 g. ofthe lter aid is added 'and the mixture filtered through an aluminumsilicate filter bed. After washing with water, the lter cake isdiscarded and the -claried beer and washings extracted with one-fourthvolume of ethyl acetate. The ethyl acetate layer is removed, theextraction repeated once and the remaining aqueous solution discarded.The combined ethyl acetate extracts are dried over sodium sulfate andthen the ethyl acetate distilled oil in vacuo keeping the temperature ofthe residue below about 30 C. The desired antibiotic is extracted fromthe brown residue with small portions of ether, a total of about 450 cc.being used.

solvent, removing any gumlike material which The'combined ether extractis poured through umn used in this step may be relatively short but itshould contain about 15 g. of aluminum oxide per 100 cc. of etherextract. After washing the column with about 1250 cc. of ether to removeany remaining antibiotic, the eiliuent ether solutions are combined andthe ether evaporated in vacuo. The residue is extracted repeatedlywith atotal of about 2200 cc. of distilled water 'and the insoluble residuediscarded. The aqueous extracts are combined, extracted with two onehalfvolume portions of petroleum ether and the petroleum ether extractsdiscarded. Alternatively, water can be added to the ether solutionobtained after passage over the adsorption column, the ether distilledfrom the two phase mixture and the residual aqueous solution extractedwith petroleum ether. The aqueous layer or extract obtained by either ofthe foregoing methods is concentrated in vacuo to the point ofcrystallization, the concentrate cooled and the white crystalline(l)\//-1pnitrophenyl 2 dichloroacetamidopropane-1,3-diol collected.About 1.54 g. of antibiotic melting in the neighborhood of 144-5 C. isobtained at this point.

A further quantity of the desired productv can be recovered from themother liquors by extracting four times with an equal volume of ether,removing the ether in vacuo, extracting the residue repeatedly withboiling methylene dichloride and concentrating the methylene dichlorideextracts to the point of crystallization. The methylene dichlorideextract is cooled to about C. and the desired (l) -p-l-p-nitrophenyl-Z-dichloroacetamidopropane 1,3 diol collected. About 0.18 g. of productmelting at about 145 C. is recovered in this manner.

After one or two recrystallizations of the above materials frommethylene dichloride, ethylene 8 ether-free aqueous solutionconcentrated to obtain the crystalline antibiotic. ,Still' anothermethod for the isolation of the antibiotic involves adsorbing theantibiotic present in the clariiied beer onto activated carbon andeluting it with ether or 80% acetone.

Example 2 A mixture consisting of 144. g. of maltose, 72

g. of distillers solubles, 72 g. of hydrolyzed casein, y

72 g. of sodium chloride and sufficient tap water to bring the volume of14,400 cc. is adjusted to pH 7.5-7.7 with 10 N sodium hydroxide solutionand dispensed in 300 cc. portions into fortyeight wide-mouth l-literErlenmeyer ilasks. The flasks are capped with two layers of gauzecottonmilk filter discs and the caps secured in place with spring clips. Theflasks are placed in an autoclave and sterilized at 121 C. for 20minutes.` After cooling, the ilasks are opened and seeded with 5 cc. perilask of a three-day old shaker ilask culture of Streptomycesvenezuelae. The inoculated flasks are capped and then incubated forthree days at 22 to 24 dichloride or ether-petroleum ether mixture the vpure (l) p 1 p-nitrophenyl-Z-dichloroacetamidopropane-1,3-diol isobtained in the form of colorless needles or elongated plates melting at15G-151 C. (uncorrected) and having an optical rotation, lalnzf, of 25.5in ethyl acetate and +18 in ethanol. Analysis of the product has giventhe following results: C, 41.0%; H, 3.74% N, 8.64% and organic Cl,21.71%. On ultraviolet absorption spectrum analysis this productexhibits a. Amax at 278 ma and an Eurem. of 312 in 0.1 N hydrochloricacid.

It will, of course, be appreciated by those skilled in the art thatvarious modiiications may be made in the above described method forcultivating the organism and isolating the pure, crystalline antibioticfrom the crude culture liquid. For example, in the isolation procedureother solvents such as amyl acetate cyclohexanone, butanol or methylisobutyl ketone may be substituted forthe ethyl acetate; nitromethanefor the ether; and benzene or dichloroethyl ether for the petroleumether. The ethyl acetate extraction of the clarified beer may be carriedout at a pH-of about 9 instead of at about pH 2 in which case it is notnecessary to pass the subsequently obtained ether solution of theantibiotic over van aluminum oxide absorption column. The ether solutionaccording to this modiiication, is extracted with dilute acid, wateradded to the ether layer and the ether evaporated from the mixture. Thegum which separates from the solution is removed and the C. on a rotarytype shaking machine (150 R. P. M.; radius of circle, 2 inches).

12 liters of the culture (pH 7.39) which contains a total of 600 mg. ofthe desired antibiotic is acidied to pH 2 by the addition of 190 cc. of3 N hydrochloric acid. 304 g.` of an aluminum silicate filter aid isadded and the suspension stirred 'for 30 to 60 minutes. An additional304 g. of filter aid is added and the mixture iiltered through a 50 g.pad of aluminum silicate. The filter cake is washed with 1212 cc. ofdistilled Water and discarded. The combined filtrate and washings isextracted by stirring for 10 minutes with one-fourth volume of ethylacetate. The ethyl acetate layer is removed and the extraction Vrepeatedonce. The aqueous layer is discarded `and the combined ethyl acetateextracts dried over 50 g. of anhydrous sodium sulfate at The sodiumsulfate is removed by filtration and the ethyl acetate distilled off invacuo at a bath temperature of 30 C. The brown residue is extracted withsmall portions of diethyl ether, a total of 300 cc. being used. Theether extract contains 504 mg. or 84% of the antibiotic present in theculture.

The ether extract is divided into three portions and each poured througha 15 g. column (6.5" x 0.75") of aluminum oxide which has previouslybeen adjusted to pH 4.7 with hydrochloric acid. 120 cc. of diethylether, in 20 cc. portions is percolated through each column. Thereceivers under the columns are changed in order to collect peatedlywith distilled water, a total of 625 cc.

being used. The combined aqueous extracts are shaken with one-halfvolume of petroleum ether and the extraction repeated once. Thepetroleum ether `.extracts are discarded and the aqueous phaseconcentrated in vacuo at a bath temperature of 30 C. to the point ofcrystallization (about 20 cc. 'I'he solution is cooled overnight at 5C., the crystals collected, washed with a small amount of cold water anddried over calcium chloride in vacuo; M. P. 144.5-145" C.;

[alge-25.5

in ethyle acetate.

Further quantities of the desired (1)-gl/-1-pnitrophenyl-2dichloroacetamidopropane 1,3 diol can be recovered from the motherliquors as described in Example 1. The crystalline product obtained asdescribed above may be puried further,1 if desired, by recrystallizationfrom methylene or ethylene dichloride; M. P. 150- l C.

What I claim as my invention is:

1. In a process for obtaining (1)-1l/-1-p-nitrophenyl-2dichloroacetamidopropane 1,3 diol, the method of isolating id diol fromculture liquids containing the sam which comprises-.extracting saidculture liquid below about pH 10 with an organic solvent having asolvent-water distribution coeiicient for the diol of over about .9,removing the organic solvent from the extract,

extracting the residue with an organic solvent having a solvent-waterdistribution coeiiicient for the diol between about 1 and 9, washing theextract with dilute mineral acid, adding water to the organic extractand evaporating off the organic solvent, extracting the aqueous solutionwith an organic fat solvent in which the diol is substantiallyinsoluble, evaporating the aqueous phase to the point of crystallizationand recovering the diol from the concentrate.

2. In a process for obtaining (l)\l/1p-nitro phenyl-2dichloroacetamidopropane 1,3 diol, the method of isolating said diolfrom culture liquids containing the same which comprises extracting saidculture liquid below about pH 10 with ethyl acetate, removing the `ethylacetate from the extract, extracting the residue with diethyl ether,washing the extract with dilute mineral acid, adding water to the etherextract and evaporating off the ether, extracting the aqueous solutionwith petroleum ether, evaporating the aqueous phase to the point ofcrystallization and recovering the diol from the concentrate.

3. In a process for obtaining(1)-1/1-1-p-nitrophenyl-2-dichloroacetamidopropane1,3diol, the method ofisolating said diol from culture liquids containing the same whichcomprises extracting said culture liquid below about pH 10 with anorganic solvent having a solvent-water distribution coeiiicient for thediol of over about 9, removing tlie organic solvent from the extract,extracting the residue with an organic solvent of the class consistingof lower dialkyl ethers and ethylenevdichloride, passing the extractover an aluminum oxide type adsorption column, adding water to theeiiiuent and evaporating oil. the organic solvent, extracting theaqueous solution with an organic fat solvent in which the antibiotic issubstantially insoluble, evaporating the aqueous phase to the point ofcrystallization and recovering the diol from the concentrate.

4. In a process for obtaining(1)-50-1-p-nitrophenyl-2-dichloroacetamidopropane-1,3-diol, the methodof isolating said diol from culture liquids containing the same whichcomprises extracting said culture liquid below about pH 10 with ethylacetate, removing the organic solvent from the extract, extracting theresidue with ether, passing the extract over an aluminum oxide type 10 yadsorption column, adding water to the eiiiuent and` evaporating oil!the ether, extracting the aqueous solution with petroleum ether,evaporating the aqueous phase to the point of crystallization andrecovering the diol from the concentrate.

5. In a process for obtaining (1)'-4/-1-p-'nitromethod of isolating saiddiol from culture liquids containing the same which comprises extractingsaid culture liquid below about pH 10 with an organic` solvent having asolvent-water distribution coeiiicient for the diol between about 1 and9, washing said extract with dilute mineral acid, adding waterto theorganic extract and evaporating oil the organic solvent, extracting theaqueous solution with an organic solvent in which the diol issubstantially insoluble, evaporating Vthe aqueous phase to the point ofcrystallization and recovering the diol from the concentrate.

6. In a process for obtaining(1)-30-1-p-nitrophenyl-2-dichloroacetamidopropane-1,3-diol, the

method of isolating said diol from culture liquids containing the samewhich comprises extracting said culture'liquid below about pH l0 withdiethyl ether, washing said extract with dilute mineral acid, addingwater to the ether extract and evaporating off the ether, extracting theaqueous solution with petroleum4 ether, evaporating the aqueous phase tothe point of crystallization and recovering the diol from theconcentrate. l

7, In a process for obtaining(1)-1/1-1-p-nitrophenyl-2-dichloroacetamidopropane-1,3-diol, the methodof isolating said diol from culture liquids containing the same whichcomprises extracting said culture liquid below about pH l0 with anorganic solvent of the class consisting of lower dialkyl ethers andethylene dichloride, passing theextract over an aluminum oxide typeadsorption column, adding water to the eliiuent and evaporating 0E theorganic solvent, extracting the aqueous solution with an organic fatsolvent in which the antibiotic is substantially insoluble, evaporatingthe aqueous phase to the-point of crystallization and recovering thediol from the concentrate.

8. In a process for obtaining(1)-[1-1-p-nitrophenyl-2-dichloroacetamidopropane-1,3-dio1, the methodof isolating said diol from culture liquids containing the same whichcomprises extracting said culture liquid below about pH 10 with diethylether, passing the extract over an aluminum oxide type adsorptioncolumn, adding water to the eliluent and evaporating off the ether,extracting the aqueous solution with petroleum ether, evaporating theaqueous phase to the point of crystallization and recovering the diolfrom the concentrate.

QUENTIN R. BARTZ.

REFERENCES CITED The following references are of record in the iile ofthis patent:

UNITED STATES PATENTS Number Name Date 1,865,880 Oberlin July 5, 19322,103,266 Lott Dec. 28,1937

